Sugar-derived oxazolone pseudotetrapeptide as γ-turn inducer and anion-selective transporter

The intramolecular cyclization of a C-3-tetrasubstituted furanoid sugar amino acid-derived linear tetrapeptide afforded an oxazolone pseudo-peptide with the formation of an oxazole ring at the C-terminus. A conformational study of the oxazolone pseudo-peptide showed intramolecular C=O···HN(II) hydrogen bonding in a seven-membered ring leading to a γ-turn conformation. This fact was supported by a solution-state NMR and molecular modeling studies. The oxazolone pseudotetrapeptide was found to be a better Cl−-selective transporter for which an anion–anion antiport mechanism was established.


S6
The tetrapeptide 7 (0.5g, 0.4171 mmol) was dissolved in 2 mL of THF/MeOH/H2O (3:1:1) and LiOH (0.042 g, 1.04 mmol) was added slowly at room temperature with continuous stirring for 3h, with the completion of reaction methanol was concentrated in vacuo and the residual solution was acidified by using 1 N HCl. The generated acid was extracted with ethyl acetate (3 × 10 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated in vacuo to give 0.45 g (92%) of the azido-acid tetrapeptide. The resulting azido-acid tetrapeptide (0.45 g, 0.379 mmol, crude) was subjected for hydrogenation in presence of H2, 10% Pd/C in methanol with continuous stirring at room temperature for 3h. After completion of reaction the reaction mixture was filtered through celite using sintered funnel. The resulting solution of methanol was concentrated in vacuo to get crude amino-acid tetrapeptide 9 as foamy solid (0.44 g, 0.379 mmol). The crude amine-acid tetrapeptides 9 (0.44 g, 0.379 mmol) were dissolved in dry CH2Cl2 with high dilution (≈0.005 M) under inert atmosphere (nitrogen gas) and Et3N (0.076 g, 0.759 mmol, 2 equiv), CMPI (0.11 g, 0.43 mmol, 1 equiv) was added at rt. Reaction mixture was stirred for 5h at 55 °C temperature. The reaction mixture was diluted with EtOAc (20 mL) and organic layer was washed with 1 N HCl (10 mL) and saturated aqueous NaHCO3 (10 mL) and brine. The organic layer was dried over Na2SO4. The solvent was removed in vacuo to give the crude product that was purified by column chromatography to afford oxazolone

S8
Oxazolone pseudotetrapeptide acetate 2b: To a mixture of 2a (0.04 g, 0.034 mmol) and pyridine (0.0056 mL, 0.068 mmol) in DCM (3 mL) at 0 o C under nitrogen atmosphere was added acetic anhydride (0.034 g, 0.035 mmol) and the reaction was allowed to attain room temperature and stirred for 12 hours. With the completion of reaction, the solvent was removed under reduced pressure to give the crude product that was purified by column chromatography to afford N-acetylated oxazolone pseudotetrapeptide 2b as a white solid (0.034 g, 84%), Rf = 0.

S22
The time course of HPTS fluorescence emission intensity, Ft was observed atλem = 510 nm (λex = 450 nm). 20 µL of 0.5 M NaOH was added to the cuvette at t = 20 s to make the pH gradient between the intra and extra vesicular system. Transporter compound 2a were added at t = 100 s and at t = 300 s, of 10% Triton X-100 (25 µL) was added to lyse vesicles for the complete destruction of pH gradient. All fluorescence time-dependent spectra were normalized between 0 (at 96 s) and 100 s (at 320 seconds) by using Equation S1. Then the time of transporter addition was adjusted to t = 0 s. Cation selectivity was checked by varying the extravesicular chloride salts (MCl) of different alkali metal cations (Fig. S13A). The difference in rate observed for different cation (Li + , Na + , K + , Rb + , Cs + ) is characteristics of cation selectivity of Ion transporter. Anion selectivity was evaluated by varying the extravesicular sodium salts of different halides (NaX) ( Figure S13B). In these assays, NaCl, NaBr, and NaI were used. The difference in transport activity was observed due to the competitive transport of X  /OH  .

Ion transport activity by Lucigenin assay:
Preparation of EYPC-LUVsLucigenin for concentration dependent assay and symport  between intra-and extravesicular system, followed by the addition of transporter at t  100 s.
Finally, vesicles were lysed by addition of 10% Triton X-100 (25 L) at t  300 s for the complete destruction of chloride gradient.
The time-dependent data were normalized to percent change in fluorescence intensity using Equation S3: where, I0 is the initial intensity, It is the intensity at time t, and I ∞ is the final intensity after addition of Triton X-100. Then the time of transporter addition was adjusted to t = 0 s.  Subsequently, the ion transport was monitored after the addition of the transporter 2a (20 µM).

Cation selectivity assay across EYPC-
For monitoring the ion transport in the presence of valinomycin, valinomycin (1 µM) was added prior to the addition of the transporter 2a.
The remarkable enhancement in ion transport activity of 2a in the presence of valinomycin gave a direct experimental insight of antiport mechanism of ion transport.

Preparation of EYPC-LUVslucigenin:
The vesicles were prepared by the following protocol as stated above. Geometrically optimized models of 2a, 2b and 9: The geometrically optimized models of 2a, 2b and 9 showed small structural changes with respect to helical pitch length. The distance between C=ON(II) is 3.18 Å in 2a, 3.29 Å in 2b and 3.43 Å in 9. The distance between Cα1Cα4 is 9.67 Å in 2a, 9.64 Å in 2b and 9.84 Å in 9.

S27
Similarly, the distance between N1C4 is 9.44 Å in 2a, 9.51 Å in 2b and 10.47 Å in 9. This suggests that compound 9 is slightly elongated helix than compound 2a and 2b which supports the compact helical architecture for 2a and 2b due to the presence of oxazolone ring leading to γturn conformation as shown in Figure 18. Figure S18: Geometrically optimized models of 2a, 2b and 9 with distance measurements.